Bone mineralization is initiated by matrix vesicles (MVs), cell-derived structures released into the extracellular matrix which are nucleation sites for hydroxyapatite (HA) formation. It is suggested that annexins are mineralization-stimulating membrane proteins that exhibit ion channel activity and facilitate the influx of Ca2+ into MVs. The process is also regulated via enzymatic degradation of inhibitory pyrophosphate by tissue-nonspecific alkaline phosphatase (TNAP). Another layer of control is exerted by circulating, mineralization-inhibiting protein fetuin-A.
The objective of our study was to examine the roles of annexins and fetuin-A in MVs function during physiological and pathological mineralization of bones.
We used two human cell lines: osteoblastic hFOB1.19 and osteosarcoma Saos-2. These cells were stimulated for mineralization for 7 and 14 days by ascorbic acid and β-glycerophosphate treatment. We compared cell morphology, intracellular distribution of proteins and formation of HA in control and levamisole (TNAP inhibitor)- or K-201 (a calcium channel inhibitor)-treated cells. We detected calcium nodules by Alizarin Red-S staining of cell cultures. Levamisole blocked mineralization of hFOB and Saos-2 cells. On the other hand, K-201 did not have a significant effect on hFOB cells whereas stimulated calcium deposition in Saos-2 cells. We then isolated MVs from these cells by collagenase digestion and determined TNAP activity using pNPP as a substrate. TNAP activity in osteosarcoma cells was at least 30 times higher than in hFOB cells and reached the maximum after 25 min. Using WB method we observed differences in annexins and fetuin-A profile in MVs from resting vs stimulated cells, but the expression of annexins and fetuin-A was similar in both cell lines. Levamisole decreased the level of fetuin-A in Saos-2 cells, but did not influence the annexin level, whereas K-201 decreased the content of fetuin-A and annexins in both cell lines.
In conclusion, understanding of the role of annexins and fetuin-A as biomarkers in TNAP-regulated function of MVs may provide novel insights into the mechanisms of physiological mineralization and may help to create TNAP and calcium channels inhibitors as therapeutic strategies to prevent pathological mineralization.
14 May 2016 - 17 May 2016